FuGene 6 transfection problems - (Mar/06/2008 )

I want to know how true it is for the story that the transfection shoule be done without P/S. Did you test this rKay447 with P/S and found no good?

-genehunter-1-

I used it with different cell lines. I think 1:3 ratio in 6 well plate was teh best for me with all cell lines I used. No media change and I still see expression upto 3 days (72 hours). yeah 50 to 70% confluency.Complexing was from 30 min up to 2 hours and still working fineUSE beta gal reporter plasmi to optomize your treansfectiondon't change media

-anwar_mt-

Yes. When I first started I accidentally did a transfection with the full media and about 80% of the cells (I assume the ones that were transfected) died and were floating in the media.

-rkay447-

Hey guys,

thanks for the help! I finally got it to work. The problem was apparently the reagent itself. When I got a new tube and did the EXACT same procedure, the transfection worked!!

Thanks again for the suggestions!

-labrat612-

Yes, when we use it we avoid exposure to air as much as we can and you need to follow the protocol exactly. It should work.

Bye

-anwar_mt-

QUOTE (labrat612 @ Mar 14 2008, 11:05 PM)

Hey guys,

thanks for the help! I finally got it to work. The problem was apparently the reagent itself. When I got a new tube and did the EXACT same procedure, the transfection worked!!

Thanks again for the suggestions!

Hey guys,anwar is right regarding storage...I also had problems when I received a small aliquot of Fugene6 from a collegue the first time- it was stored frozen in an eppendorf tube. Then I read on the roche-applied-science homepage the following (see below). Sometimes it would be better to first read the pack insert, then start working...

"Both the new version of FuGENE 6 Transfection Reagent and FuGENE® HD Transfection Reagent must not be stored below 0°C! Further, to achieve optimum stability of the products the vials have always to be stored very tightly closed. Never aliquot the contents for storage!Note: As a function of time, storage at low temperatures will separate active components of FuGENE 6 Transfection Reagent. Nevertheless, this effect is reversible upon warming reagent to ambient temperature and thoroughly mixing. Storage at low temperatures is not required to maintain shelf life.

• For FuGENE HD Transfection Reagent the order of pipetting must not be changed: Always dilute the reagent by pipetting directly into serum-free medium containing DNA. Do not allow the FuGENE HD Transfection Reagent to contact the plastic walls of the tube containing the serum-free medium during the dilution step."